Despite advances in water and food processing, outbreaks of disease from waterborne and foodborne pathogens still occur frequently in the United States. Bacterial water- and foodborne pathogens include Clostridium species, e.g., Clostridium botulinum (botulism) and Clostridium perfringens (food poisoning); Staphylococcus aureus and other staphylococci (food poisoning); Streptococcus species (gastroenteritis); enteropathogenic strains of Escherichia coli (gastroenteritis); Shigella species, e.g., Shigella dysenteriae (dysentery), and Shigella flexneri and Shigella sonnei (gastroenteritis); Bacillus cereus (gastroenteritis); Salmonella species, e.g., Salmonella enteritidis and Salmonella paratyphi (gastroenteritis), as well as Salmonella typhi (typhoid fever); the Salmonella-like Arizona hinshawii; Yersinia enterocolitica (gastroenteritis); Vibrio parahemolyticus (gastroenteritis); Vibrio cholerae (cholera); species of Campylobacter (gastroenteritis); species of Leptospira (leptospirosis); Brucella species (undulant fever); Francisella tularensis (tularemia); and Listeria monocytogenes (listeriosis). There are also numerous water- and foodborne protozoan pathogens, such as Entamoeba histolytica, Giardia lamblia, Cryptosporidium, Microsporidia, and Cyclospora. Many of these organisms can infect humans via either foodborne or waterborne routes (e.g., Salmonella), while others are more particularly limited to transmission through either water or food.
In an attempt to avoid disease, food and water is often sampled and tested prior to distribution to determine whether the food/water is contaminated by pathogenic organisms. Numerous testing methods are available, but the following steps (or similar steps) are common to many methods (particularly those involving bacteria rather than protozoan organisms):
First, a pre-enrichment step is performed on the sample to increase the number of pathogenic organisms present. The organisms are cultured in a non-selective growth medium, e.g., trypticase soy broth or peptone water, typically for 24 hours or more. The pre-enrichment step is disadvantageous because it is generally inconvenient to stall distribution of water or fresh foodstuffs while their samples are culturing, particularly where freshness or spoilage concerns are present or the food/water is unwieldy to store. However, pre-enrichment is usually necessary because pathogenic organisms may be present in very dilute amounts, thus making them difficult to detect.
Second, an enrichment step is performed wherein a portion of the culture medium is transferred to an enrichment medium containing inhibitors that select for a pathogen of interest. The selected pathogen will grow further while other organisms are inhibited. This step, which essentially furthers the concentration of pathogens begun by the pre-enrichment step, can also be inconvenient and time-consuming.
Third, a measurement step is performed to discern whether pathogens of interest are present. Generally, a portion of the enrichment medium is streaked onto selective/differential agar media. The media will contain inhibitors effective against most organisms except the pathogen of interest. Indicator compounds (e.g. dyes) allow pathogen types to be differentiated, and thus indicate the presence and number of pathogens of interest. Exemplary alternative measurement steps are radioimmunoassay (RIA) tests, immunofluorescent assay (IFA) tests, enzyme immunoassay (EIA) tests, DNA methods (e.g., PCR), and phage methods. IFA and DNA methods are generally used to detect protozoan pathogens in lieu of the other methods noted above.
If pathogenic organisms are detected in the food/water, the food/water can be destroyed or withdrawn prior to consumption. Alternatively, it can be treated to eliminate or substantially reduce the presence of pathogens. Filtration, pasteurization, chemical treatment (e.g., chlorination or ozonation), and radiation treatment are common methods of reducing or eliminating pathogens from food/water.
U.S. Pat. No. 3,217,982 to Wilsmann et al. illustrates a method of reducing pathogens (specifically bacterial pathogens) within beverages by use of continuous flow bowl centrifugation. As an introduction to continuous flow bowl centrifugation, an exemplary continuous flow bowl centrifuge (commonly referred to as a Latham bowl centrifuge) is shown at 20 in FIG. 1. Fluid is introduced into the end (top or bottom) of a spinning bowl 22 from an axially-directed input line 24 with length r1 located near the rotational axis of the bowl 22. As the fluid flows up the height h of the bowl 22, centrifugal force forms a density gradient in the fluid with the densest (heaviest) matter (e.g., food particles or other detritus) located near the wall of the bowl 22 and the lighter matter (e.g., water) being located closer to the rotational axis. As liquid is introduced into the bowl 22, the lighter portion of the fluid is emptied at the same flow rate from an axially-situated and axially-directed exit line 26 with length r2 which exits the end of the bowl 22 opposite the input line 24. The Wilsmann et al. invention utilizes continuous flow bowl centrifugation to remove microorganisms from beverages by heating the beverage during centrifugation, and also by providing dual exit lines, one of which is used to draw off the heavy microorganism-rich fraction for removal from the beverage and the other being used to draw off the lighter microorganism-depleted fraction for later consumption.
However, studies of continuous flow bowl centrifugation wherein contaminated fluid is centrifuged without heating have found that continuous flow bowl-type centrifugation alone is not efficient in concentrating pathogens from the water/food. Some studies of bowl-type continuous flow centrifuge for concentration of Cryptosporidium oocysts report recovery efficiency to be between 11% to 31.2%. Whitmore et al., Wat. Sci. Tech. 27 (3-4): 69-76, 1993. Goatcher et al. also tested a bowl-type continuous flow centrifuge system in the concentration of Cryptosporidium oocysts from water and reported obtaining recovery rates of between 2 to 20 times those observed with an ASTM filtration method. American Society of Microbiology Abstracts Q-212, 1995. Since Clancy et al. report that the ASTM method provides Cryptosporidium recovery rates averaging around 2.8%, such recovery rates are still well under 50%. Journal of the American Water Works Association 86: 89-97, 1994.
When it is then considered that protozoan Cryptosporidium oocysts are among the largest food/waterborne pathogens and thus should achieve a higher degree of concentration during centrifugation than smaller bacterial pathogens (in accordance with Stokes' equation), it can be expected that even lower recoveries of bacterial pathogens would result from centrifugation. As a result, even though continuous flow centrifugation is generally cheaper and less time-consuming than filtration, it has not gained acceptance as a method for concentrating pathogens from fluids and providing accurate measures of their total count therein. Owing to the importance of detecting waterborne and foodborne pathogens and preventing their transmission to the general population, it would clearly be beneficial to have an inexpensive, rapidly-performed, and accurate method available for the concentration of such pathogens.